Skin graft product

ABSTRACT

An apparatus and method for the production of substitute skin that advantageously reduces the amount of donor dermal cells needed from non-wound areas of a patient having a wound to be auto-grafted is reduced by using all of the harvested skin cells. A 3D printer is used to construct a wound graft product from the harvested skin cells without wasting any of the harvested skin cells. In a case of an irregularly shaped wound, wastage of harvested skin associated with trimming is avoided.

FIELD OF THE INVENTION

The invention relates to the medical arts, more particularly, to tissueengineering especially tissue engineering in which three-dimensionalprinting technology is used.

BACKGROUND OF THE INVENTION

Healing wounds is a complex process of tissue repair and regeneration inresponse to injury. The healing response in skin wounds attempts toreconstitute a tissue similar to the original damaged one and this isaccomplished via the concerted action of numerous skin cell types,collagens, cytokines, growth factors (GF s), chemokines, cell surfaceand adhesion molecules, as well as multiple extracellular matrixproteins. Autologous split-thickness skin grafting currently representsthe most rapid, effective method of reconstructing large skin defects;however, in cases where a significant quantity of harvested graft isrequired, it represents yet another trauma to an already injuredpatient.

Some patent literature and academic literature is mentioned as follows,generally in roughly chronological order:

Wille, Jr., “Method for the formation of a histologically-complete skinsubstitute,” U.S. Pat. No. 5,292,655 issued Mar. 8, 1994;

Bernard, et al., “Process for creating a skin substitute and theresulting skin substitute,” U.S. Pat. No. 5,639,654 issued Jun. 17,1997;

Bernard et al., “Skin substitute,” U.S. Pat. No. 5,667,961 issued Sep.16, 1997;

Wille, Jr., “Serum free medium for use in the formation of ahistologically complete living human skin substitute,” U.S. Pat. No.5,686,307 issued Nov. 11, 1997;

Takai et al., “Wound healing composition using squid chitin and fishskin collagen,” U.S. Pat. No. 5,698,228 issued Dec. 16, 1997;

Wille, Jr., “Cell competency solution for use in the formation of ahistologically-complete, complete, living, human skin substitute,” U.S.Pat. No. 5,795,781 issued Aug. 18, 1998;

S. Hybbinette et al., “Enzymatic dissociation of keratinocytes fromhuman skin biopsies for in vitro cell propagation,” Exp Dermatol., 1999:Feb; 8(1):30-8;

Mares-Guia, “Non-immunogenic, biocompatible macromolecular membranecompositions, and methods for making them,” U.S. Pat. No. 6,262,255issued Jul. 17, 2001;

D. W. Hutmacher, “Scaffold design and fabrication technologies forengineering tissues—state of the art and future perspectives,” J.Biomater. Sci. Polymer Edn, 12:1, 107-124 (2001);

Conrad et al., “Skin substitutes and uses thereof,” US 20020164793published Nov. 7, 2002;

Ramos et al., “Method for the preparation of immunologically inertamniotic membranes,” US 20040126878 published Jul. 1, 2004;

Conrad et al., “Skin substitutes and uses thereof,” U.S. Pat. No.6,846,675 issued Jan. 25, 2005;

Conrad et al., “Skin substitutes and uses thereof',” US 20050226853published Oct. 13, 2005;

S. G. Priya, et al., “Skin Tissue Engineering for Tissue Repair andRegeneration,” Tissue Engineering: Part B, 14:1, 2008, 105-118;

G. S. Schultz, et al., “Interactions between extracellular matrix andgrowth factors in wound healing,” Wound Rep Reg 17, 153-162 (2009);

Conrad et al., “Skin substitutes and uses thereof,” U.S. Pat. No.7,541,188 issued Jun. 2, 2009;

Woodroof, “Laser-Perforated Skin Substitute,” US 20090230592 publishedSep. 17, 2009;

Woodroof, et al., “Artificial Skin Substitute,” US 20090232878 publishedSep. 17, 2009;

Woodroof; “Temporary Skin Substitute comprised of biological compoundsof plant and animal origins,” US 20090234305 published Sep. 17, 2009;

Woodroof, et al., “Skin Substitute Manufacturing Method,” US 20100000676published Jan. 7, 2010;

Woodroof, et al., “Artificial skin substitute,” U.S. Pat. No. 7,815,931issued Oct. 19, 2010;

Mirua, et al., “Skin Substitute Membrane, Mold, and Method of EvaluatingExternal Preparation for Skin,” US 2011009881 5 published Apr. 28, 2011;

Israelowitz et al., “Apparatus for the growth of artificial organicitems, especially human or animal skin,” US 20110159582 published Jun.30, 2011;

Guenou, “Methods for Preparing Human Skin Substitutes from HumanPluripotent Stem Cells,” US 20110165130 published Jul. 7, 2011;

Bush et al., “Bioengineered Skin Substitutes,” US 20110171180 publishedJul. 14, 2011;

Yoo et al., “Delivery system,” US 20110172611 published Jul. 14, 2011;

M. V. Karaaltin et al., “Adipose Derived Regenerative Cell Therapy forTreating a Diabetic Wound: A Case Report,” Oct. 6, 2011;

Chernokalskaya, et al., “Polymeric Membranes with Human Skin-likePermeability Properties and uses thereof,” US 20110281771 published Nov.17, 2011;

Miura et al., “Application method of external dermatologicalmedications, evaluating method of the same, application evaluatingapparatus, and application evaluating program,” US 20120022472 publishedJan. 26, 2012;

D. Rosenblatt, “Researchers aim to ‘print’ human skin,” Feb. 15, 2011,www.cnn.com;

Miura et al., “Skin substitute membrane, mold, and method of evaluatingexternal preparation for skin,” US 20120109300 published May 3, 2012;

R. Kirsner, et al., “Spray-applied cell therapy with human allogeneicfibroblasts and kertinocytes for the treatment of chronic venous legulcers: a phase 2, multicentre, double-blind, randomised,placebo-controlled trial,” www.thelancet.com, vol. 380, Sep. 15, 2012;

B. Raelin, “Wake Forest 3D Prints Skin Cells Onto Burn Wounds,” Jul. 19,2012, www.3dprinter-world. com;

A. Lutz, “Printed Skin Cells Will Change How We Treat Burns Forever”,Aug. 03, 2012, www.businessinsider.com;

Miura et al., “Skin substitute membrane, mold, and method of evaluatingexternal preparation for skin,” U.S. Pat. No. 8,337,554 issued Dec. 25,2012;

“Printing Skin,” www.medicaldiscoverynews.com/shows/202 printSkin.html,undated;

C. M. Zelen, et al., “A prospective randomised comparative parallelstudy of amniotic membrane wound graft in the management of diabeticfoot ulcers,” International Wound Journal, ISSN 1742-4801, 2013;

H. Kim, et al., “Evaluation of an Amniotic Membrane-Collagen DermalSubstitute in the Management of Full-Thickness Skin Defects in a Pig,”Archives of Plastic Surgery, 2013, 40:1, 11-18;

“SkinPrint: 3D Bio-printed human skin can help burn victims”, May 06,2013, www.3ders.org;

K. Maxey, “3D Printed, Transplantable Skin in 5 Years?”, May 17, 2013,www.engineering.com;

H. Briggs, “Artificial human ear grown in lab,” Jul. 31, 2013,www.bbc.co.uk;

S. Leckart, “How 3-D Printing Body Parts Will RevolutionizeMedicine,”Aug. 06, 2013, www.popsci.com;

Thangapazham et al., “Hair follicle neogenesis,” US 20130209427published Aug. 15, 20131

T. Lu et al., “Techniques for fabrication and construction ofthree-dimensional scaffolds for tissue engineering,” Internat'l Journalof Nanomedicine, 2013:8, 337-350.

Although there are a number of reports of skin autografts produced invitro, they take weeks to generate—which is too long a waiting periodfor a patient whose wound needs treatment. Quicker production of skinautografts is an unmet need and unsolved problem.

In several studies conducted using amniotic membrane (AM) in both acuteand chronic wounds, much of the first round placement is absorbed intothe body. In some cases, it takes as many of 3-4 full grafts of AM inorder to result in full closure of the wound. Less graft being absorbedinto the body so that it is unable to contribute to closing the wound isan unsolved problem.

Another difficult unsolved problem has been that when an undamaged donorarea of skin of a patient is harvested and used as an autograft fortreating the patient's own wound, the donor site often becomes anon-healing wound.

There are complicated, unsolved problems and unmet needs for bettertechnologies in wound grafting and wound healing.

SUMMARY OF THE INVENTION

The invention addresses the above-described problems by processing ALLof the harvested skin cells taken from a healthy donor site on thepatient with the wound to construct a customized skin graft product tobe auto-grafted onto the wound. Production of a customized skin graftpreferably is accomplished by operation of a three-dimensional (“3D”)printer, which is supplied with substrate material and autologous skincells and “prints” the supplied skin cells onto an agar plate or othersurface.

Advantageously the amount of donor dermal cells needed from non-woundareas of a patient having a wound to be auto-grafted is reduced by usingall of the harvested skin cells. A 3D printer is used to construct awound graft product from the harvested skin cells without wasting any ofthe harvested skin cells. In a case of an irregularly shaped wound,wastage of harvested skin associated with trimming is avoided. Theinvention's provision of a skin grafting method that requires only theleast amount of precious skin of the donor site to be damaged is highlyimportant given major functions of skin: acting as a protective barrierfrom environmental insults including trauma, radiation, harshenvironmental conditions and infection, providing thermoregulation(through sweating, vasoconstriction or vasodilation) and controllingfluid loss. This minimization of skin damage provided by the invention,in addition to the ability to continually regenerate the necessary skinuntil healing is complete, represent major advances in wound care.

A major objective of the invention is to use the patient's own skillcells to re-create a strong, persistent organ replacement solution.

The invention in a preferred embodiment provides a computerized skinprinting system, comprising: a quantity of living donor skin cellsharvested from a non-wound area of a patient having a to-be-treatedwound or tissue defect; a three-dimensional printer that processes thequantity of living donor skin cells harvested from a non-wound area of apatient having the wound or tissue defect, wherein the three-dimensionalprinter is under control of a controller connected to thethree-dimensional printer; an imaging device (such as, e.g., an imagingdevice that comprises a camera; an imaging device that comprises a videocamera; an imaging device that comprises a hand-held device; an imagingdevice that is movable to be positioned relative to the wound beingimaged; etc.); and a computer that performs steps of receiving a set ofimages (such as, e.g., a set of one wound image; a set of multipleimages) taken by the imaging device of the wound or tissue defect andprocessing the imaged wound or tissue defect into a set of skin-printinginstructions that are provided to the controller connected to thethree-dimensional printer; such as, e.g., a skin printing system furthercomprising a sizing grid that is projected onto the wound or tissuedefect while the imaging device is being operated; a skin printingsystem further comprising a monitor connected to the computer; a skinprinting system further comprising a keyboard connected to the computer;a skin printing system further comprising at least one syringe pump(such as e.g., a syringe pump that contains the quantity of living donorskin cells harvested from the non-wound area) under control of thecontroller; a skin printing system further comprising a surface ontowhich the three-dimensional printer prints a skin product (such as,e.g., a skin printing system wherein the skin product printed onto thesurface corresponds to a model generated by the computer from the set ofwound images); a skin printing system further comprising a pump, andwherein skin cells in a syringe are pumped by the pump into thethree-dimensional printer; a skin printing system wherein the computerdigitizes a wound image and models the digitized image into a set ofprinting instructions; a skin printing system further comprising an agarplate comprising the surface onto which the three-dimensional printerprints the skin product; a skin printing system wherein thethree-dimensional printer is supplied with both a quantity of livingskin cells from the patient with the imaged wound and a quantity ofmaterial not from the patient with the imaged wound (such as, e.g.,collagen or another scaffold-building material as the non-patientmaterial); a skin printing system further comprising a digitizer; andother inventive skin printing systems.

In another preferred embodiment, the invention provides an autografttreatment method of a wound of a patient, comprising: preparing thewound to be imaged; imaging the wound to obtain a set of images (suchas, e.g., a wound imaging step that comprises photographing the wound);based on the set of images of the wound, modeling (such as, e.g.,three-dimensional modeling) a skin graft product, wherein the modelingis performed by a computer, processor, or other machine; harvestingdermal cells from a donor site of the patient; from the harvested dermalcells, preparing a live cell suspension; loading a plate into athree-dimensional printer (such as, e.g., a printer-loading step thatcomprises loading an agar gel plate onto a platen of the printer);constructing a scaffold onto the plate (such as, e.g., ascaffold-constructing step in which the scaffold is constructed usinglittle or none of the live cell suspension; a scaffold-constructing stepthat comprises constructing a scaffold of collagen (such as, e.g.,bovine collagen (such as, e.g., Bovine Collagen Type 1)); etc.); seedingthe scaffold with cells from the live cell suspension, until the modeledskin graft product has been constructed; when the skin graft product hasbeen constructed, removing the skin graft product from the printer andfrom the plate; and after the removing step, placing the skin graftproduct in the wound; such as inventive methods wherein in theharvesting step, an amount of dermal cells harvested is in approximatelya 1:5 ratio of skin harvested to skin estimated to be needed to treatthe wound by conventional skin grafting; inventive methods wherein inthe harvesting step, a maximum size is a 4 cm² split-thickness graftusing standard dermatome techniques; inventive methods wherein in theharvesting step, an amount of dermal cells harvested is does not exceeda 1:5 ratio of cells harvested to cells estimated to be needed to treatthe wound by conventional skin grafting; inventive methods comprisingdissociating and culturing the harvested cells in a culture medium (suchas methods comprising adding allogeneic fibroblasts and keratinocytes tothe culture medium); inventive methods further comprising securing theskin graft product with sutures and covering the skin graft product witha bandage; inventive methods comprising constructing multiple skin graftproducts for a same wound; inventive methods comprising constructing afirst skin graft product and a second skin graft product for a samewound, on different days; a method further comprising printing insulininto the skin graft product being constructed; a method furthercomprising printing or spraying amniotic membrane into the skin graftproduct being constructed; and other inventive methods.

In another preferred embodiment, the invention provides a skin graftproduct constructed from skin cells of a patient having a wound, whereinan amount of patient skin cells is less than the patient skin cells thatwould be estimated to be needed to treat the wound if only the patientskin cells were used, such as, e.g., an inventive skin graft productconsisting of: an amount of patient skin cells which is less than thepatient skill cells that would be estimated to be needed to treat thewound by conventional skin grafting if only the patient skin cells wereused; and an amount of material other than patient skin cells; aninventive skin graft product wherein the amount of patient skin cells isselected from the group consisting of: about ⅔ what would be estimatedto be needed to treat the wound if only the patient skin cells wereused; less than ⅔ what would be estimated to be needed to treat thewound if only the patient skin cells were used; less than ½ what wouldbe estimated to be needed to treat the wound if only the patient skincells were used; less than ⅓ what would be estimated to be needed totreat the wound if only the patient skin cells were used; less than ¼what would be estimated to be needed to treat the wound if only thepatient skin cells were used; and less than ⅕ what would be estimated tobe needed to treat the wound if only the patient skin cells were used;an inventive skin graft product wherein the amount of material otherthan patient skin cells comprises one or more of bovine collagen, growthfactors, amniotic membrane and cytokines; and other inventive skin graftproducts.

In another preferred embodiment, the invention provides a method oftreating a patient wound, comprising: constructing a set of custom skingraft products G1 . . . Gn customized to the wound; placing the customskin graft product G1 onto the wound; and placing the custom skin graftproduct Gn onto the custom skin graft product Gn−1 already placed on thewound, such as, e.g., an inventive method comprising layering customskin graft products onto the wound over a period of days; and otherinventive methods.

The invention in another preferred embodiment provides a method ofavoiding wastage of dermal cells harvested for autografting to treat awound of a patient, comprising: harvesting a quantity of skin cells froma non-wound site of the patient having the wound; processing all of theharvested quantity of skin cells into an autograft skin product withoutwasting or discarding any of the harvested quantity of skin cells (suchas, e.g., a processing step that comprises three-dimensional printing ofan irregular three-dimensional shape); and applying the autograft skinproduct onto the wound; such as, e.g., inventive methods furthercomprising meshing the autograft; inventive methods wherein a ratio ofsurface area of the wound to surface area of a harvest site is about 5square inches of wound to 1 square inch of harvest site, which isexpressed as a Wound/Harvest Areas Ratio of 5:1; inventive methodswherein a Wound/Harvest Areas Ratio is in a range of from 2:1 to 7:1;inventive methods wherein the Wound/Harvest Areas Ratio is in a range offrom 5:1 to 7:1; and other inventive methods.

The invention in another preferred embodiment provides an auto-graftingmethod for treating a wound of a patient, comprising: harvesting aquantity of skin cells from a patient; and auto-grafting onto the woundof the patient the quantity of harvested skin cells, with the quantityof autografted harvested skin cells being substantially equal to thequantity of harvested skin cells (such as, e.g., an auto-grafting stepthat comprises auto-grafting a three-dimensional irregularly-shaped skingraft product); an auto-grafting method further comprising constructing,via operation of a three-dimensional printer, a skin graft productcomprising the quantity of harvested skin cells; and other inventiveauto-grafting methods.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a diagram of a computerized skin printing system in anembodiment of the invention.

FIG. 2 is a diagram of an inventive method of producing an inventiveautograft product, in an embodiment of the invention.

FIG. 3 is a diagram of steps in an inventive autologous grafting method.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT OF THE INVENTION

In a dermal autograft that comprises a quantity of harvested patientdermal cells, the invention advantagously minimizes the quantity ofharvested patient dermal cells that are needed for an autograft to covera particular wound. For such minimization, harvested patient dermalcells (preferably ALL of the harvested patient dermal cells) are used incombination with a quantity of material which is NOT harvested patientdermal cells, to construct a dermal autograft product to be applied to awound. Preferred construction methods for use in the invention are,e.g., a layering method performed by a 3D printer (such as, e.g., 3Dprinter 1 in FIG. 1); a method in which a computerized skin printingsystem is used (such as a computerized skin printing system of FIG. 1,see Example 1 herein); etc.

A preferred example of material which is NOT harvested patient dermalcells and which is useable in the invention is collagen, such as, e.g.,Bovine Collagen Type I; Collagen IV; etc. As to Collagen IV, see, e.g.,M. Paulsson, “Basement Membrane Proteins: Structure, Assembly, andCellular Interactions,” Critical Reviews in Biochemistry and MolecularBiology, 27(½): 93-127 (1992).

The inventive methodology preferably is used to fabricate then printskin tissue using much smaller areas of donor skin (such as, e.g., nolarger than 4 cm² split-thickness grafts harvested using standarddermatome techniques) compared to conventional methodology. Theinvention's provision of the ability to use such smaller areas of donorskin corresponds to a significant reduction in skin injury andsubsequently less opportunity for transformation into a chronic wound orother sequelae common to donor sites. Advantageously, the inventionprovides an improved ratio of wound area to donor site (such as a 5:1ratio of wound area to donor site; a 6:1 ratio of wound area to donorsite; a 7:1 ratio of wound area to donor site; etc.) compared to agrafting methodology having a 1:1 up to 3:1 ratio of wound area to donorsite for a mesh graft. Advantageously, the invention is useable for arelatively small area of donor site to cover relatively much wound site,such as, e.g., being able to cover 5-7 times, or more, of the donorsite.

A preferred methodology of combining harvested patient dermal cells andother material which is NOT harvested patient dermal cells is for cellsfrom the respective donor site and non-donor sources to be processeduntil ready for loading into a set of dispensers in a 3D printer, andthe 3D printer is used to perform a printing process by which thepatient dermal cells and other materials are printed into a unitarygraft product.

To obtain the patient dermal cells, preferably small split thicknessskin grafts are created and epidermal cells harvested, after which theheterogeneous mixture of cell types comprising mainly fibroblasts andkeratinocytes is dissociated and cultured using standard cell culturetechniques. Preferably, to stimulate rapid proliferation, allogeneicfibroblasts and keratinocytes are added to culture media along with acocktail including appropriate growth factors.

In a preferred example of a printing process, autologous cells whichhave been incubated with allogeneic fibroblasts and keratinocytes areprinted onto a bovine collagen matrix in the size, shape, and depth ofthe patient's particular wound. In a most preferred example, collagen isprinted first, then skin cells are layered onto the collagen. Preferablythe collagen matrix is fortified with growth factors, amniotic membrane,and specific cytokines which serve as an active extracellular matrix(ECM) and basement membrane structure. Such procedures are preferred inorder to set in motion a process by which the partially autologous skingraft will mimic the architecture of the patient's own tissue.

Following preparation of the wound bed, a skin structure producedaccording to the invention is transplanted into the analogous structureof the wound.

An advantage of the invention is to use the patient's own skill cells tore-create a strong, persistent organ replacement solution.

Additionally, the time in which the replacement product is produced ismuch faster than the weeks needed to generate skin autografts producedin vitro using conventional methodology. The current state of thescience has not reported manipulating cell proliferation at the rateneeded for a 3-7 day growing phase. By contrast, advantageously, 3D cellprinting according to the invention using an enhanced cell proliferationmethod with a mixture of cell types, ECM proteins, growth factors, andcytokines greatly reduces the time for regeneration of an adequate skingraft suitable for transplantation and healing.

Unlike skin substitutes such as the dermal matrices Alloderm (humancadaveric), Strattice, or Integra (porcine sources) which are costprohibitive and can be immunoreactive, the invention advantageously isused to recreate or regenerate a patient's own skin, in the shape anddepth analogous to the injury. The resulting graft is less expensivecompared to the mentioned products and has a better chance to “take”.Addition of allogeneic cells bolster and enhance proliferation of thepatient's own fibroblasts and keratinocytes, and provide a source ofconstituents such as extracellular matrix and growth factors.

As may be further appreciated with reference to FIG. 3, an example of aninventive skin printing process is step-wise as follows:

1) Preparing 301 the wound 300 (e.g., NPWT—to manage exudate,reduce/eliminate infection, create vascularized granular bed of tissue).

2) Photographing 302 the wound 300.

3) Automatically modelling 303 the to-be-produced graft in 3D from thewound photo.

4) Obtaining 304 dermal cells from donor site (estimating a ratio, suchas estimating a 1:5 ratio).

5) Preparing 305 a live cell suspension using the dermal cells from thedonor site.

6) Loading 306 a plate (such as an agar plate) into a 3D printer (suchas by loading an agar plate onto a platen of a 3D skin printer).

7) Physically rendering 307 an acellular dermal matrix (ADM) scaffoldwith collagen (such as pre-processed Bovin Collagen Type I).

8) Seeding 308 the ADM scaffold with live cells processed from theautologous graft obtained in step 4 of this Example (step 304 in FIG.3). Note, ADM may contain allogeneic fibroblasts. This step is alsoaccomplished by “printing” the cells onto the ADM.

9) Removing 309 printed skin from the 3D printer and agar gel plate.

10) Performing a step 310 of placing the printed skin in the wound 300,securing with sutures and covering with a suitable bandage.

An inventive method of producing an inventive autograft product also canbe appreciated with reference to FIG. 2. Surgical instrument 18 is usedto separate epidermis 19 from skin at a donor site preferably of a samepatient who has wound 17 (FIG. 1).

Separated epidermis 19 is processed 200 by enzymatic cell separation toproduce separated dermal cells 19A which are dissolved 201 to produce adermal cell solution 19B.

Dermal cell solution 19B is cultured 202 onto plates to provide plateddermal cells 19C and/or is split 203 into dermal cell solutions 19D(such as 70% confluency).

Cultured dermal cells 19C and dermal cell solutions 19D are harvested204, 205 to be transferred to 3D printer cell dispensers such asdispenser 20.

Examples of contents of 3D printer cell dispenser 20 are, e.g.,autologous fibroblasts, keratinocytes, ECM proteins, growth factors (GFs), cytokines. Examples of contents of 3D printer cell dispenser 21 are,e.g., GF, insulin, PDGF, eNOS. Examples of contents of 3D printer celldispenser 22 are lyophyllized amniotic membrane.

A 3D printer (such as 3D printer 1 of FIG. 1) prints 206 the contents ofthe dispensers 20, 21, 22 onto a substrate 23 to produce a culturedgraft preferably comprising bovine collagen, media, growth factors (GFs), etc.

Optionally an electrical field 207 is applied in a region of thesubstrate 23 during printing 206.

It will be appreciated that printing 206 from dispensers 20, 21, 22 isnot required to be performed simultaneously and that printing 206 may beperformed in various sequences.

An example of harvesting grafts is to harvest a first graft at 7 days(from when the epidermis was removed from the donor site), and tomaintain other grafts unharvested for a period of time until neededthrough final closure.

The invention may be further appreciated with reference to the followingexamples, without the invention being limited thereto.

EXAMPLE 1

In one inventive example, as may be appreciated with reference to FIG.1, an inventive computerized skin printing system comprises athree-dimensional (3D) printer 1. Preferably the 3D printer 1 is cooledor temperature-controlled. An example of a 3D printer 1 is a 3D printercapable of printing living cells. The 3D printer 1 comprises at leastone dispenser head 2 from which emerges cells that are being printedonto a surface 3 (such as, e.g., an agar plate) which is accommodated ona platen 4 within the 3D printer. The dispenser head 2 is attached toprint head 5 which is positionable in (x, y, z) dimensions, whichpositioning is controlled by controller 6. Controller 6 also controls asyringe pumping system 7.

Syringe pumping system 7 comprises syringe 8 in which is contained skincells harvested from the patient for whom the auto-graft product isbeing made and syringe 9 in which is contained material which does NOTinclude the patient's skin cells, such as, e.g., bovine collagen;allogeneic skin cells; etc. System 7 optionally comprises static mixers.Syringes 8, 9 supply the 3D printer 1 via tubes 8A, 9A respectively.Components used by the 3D printer to print an auto-graft skin productare pumped from syringes 8, 9 to the dispenser head 2.

Controller 6 is electrically connected by electrical connection 10 tothe 3D printer 1 and by electrical connection 11 to the pumping system7.

Controller 6 is electrically connected via data line 12 to a computer13. As an example of computer 13 is a computer comprising a digitizer,the computer having software loaded thereon such as, e.g., software thatdigitizes an image of a wound and models the defect for printing;software that digitizes an image of a wound and automatically detectswound boundaries and models the defect for printing; etc. In someembodiments, wound boundaries are manually detected. Computer 13receives human operator input via an input device 14 which in FIG. 1 isillustrated as a keyboard but is not necessarily limited to a keyboard.A human operator reviews output from computer 13 on a monitor 15.

Components illustrated separately in FIG. 1, such as, e.g., input device14 and monitor 15, are not necessarily required to be separate physicalstructures and can be integral with each other. Also, in FIG. 1, cablesor connecting lines that are illustrated are not necessarily required inall embodiments to be physical structures and in some embodiments awireless connection is provided.

Computer 13 is connected to an imaging device 16 such as, e.g., acamera. Preferably imaging device 16 delivers video images to computer13. Imaging device 16 is positionable to image a wound on a livingpatient, such as, e.g., being positionable via a stable structure suchas an articulated arm, tripod, cart or frame. Imaging device comprises acomponent 16A (such as, e.g., a lens) which in operation is positionedin a direction of a wound or other tissue defect 17. Preferably a sizingguide (such as, e.g., a sizing grid) is provided in a region of thewound 17 (such as, e.g., a laser grid for sizing) while the imagingdevice 16 is imaging the wound 17. Preferably a laser sizing grid isprojected onto and/or near the wound 17 to provide data for sizing thewound. In another embodiment, graticulated markers are positionedproximate the wound to provide sizing information to the imaging device16.

Preferably computer 13 performs steps of receiving a set of images takenby the imaging device 16 of the wound or tissue defect 17 and processingthe imaged wound or tissue defect into a set of skin-printinginstructions that are provided to the controller 6 connected to the 3Dprinter 1.

The system of FIG. 1 is useable to process a quantity of living donorskin cells harvested from a non-wound area of a patient having the woundor tissue defect 17.

EXAMPLE 2

Application of the dissociated cells and other agents by the 3D printer,specifically, the configuration of the celldispenser/applicator/syringe/air-brush, is dependent upon the type anddepth of the wound. The number of “layers” or “passes” the celldispenser must take with each agent applied to the collagen matrix inthis Example is at least one layer.

This approach of layering the patient's own fibroblasts, keratinocytes,etc., with commercially available amniotic membrane, growth factors,etc., is used to manipulate the healing process through woundsupplementation with agents that are natural contributors to the woundhealing process and specifically crucial for each particular wound type.

EXAMPLE 3

Examples of techniques are as follows.

Example 3.1

Following harvest of the donor site, individual cells of the epidermallayer are dissociated from the dermis. Dissociation of skin cells isaccomplished by traditional trypsin: EDTA methods which is a preferablemethod for isolating keratinocytes from human skin. Human serum, bovineserum albumin, serum fibronectin, type IV collagen, and laminin added totraditional cell culture media provide support to the fibroblasts andkeratinocytes. These basement membrane protein constituents form thelayers of the extracellular matrix on which these epidermal and dermalcells grow. They are present in every tissue of the human body. They arealways in close apposition to cells and it is well known that they notonly provide structural support in the form of an organized scaffold,but they also provide functional input to influence cellular behaviorsuch as adhesion, shape, migration, proliferation, and differentiation.Disassociated cells are incubated and continually shaken in cell cultureflasks at 37° C. Cells are sub-cultured prior to confluency and allowedeither to continue to proliferate in dissociated cell suspension flasks,plated on collagen plates to continue growth, or plated via the skinprinter onto bovine collagen substrates.

Example 3.2

In this Example, a bovine collagen matrix is augmented with growthfactors such as Platelet-Derived Growth Factor (PDGF), epidermal NitricOxide Synthase (eNOS), Vascular Endothelial Growth Factor (VEGF), andTumor Necrosis Factor Beta (TNF-beta). Low-dose insulin is added to alsopromote cell growth and proliferation. Insulin is a powerful growthfactor that has been used in animal and human clinical trials of woundhealing. Insulin has been used as a topical agent to accelerate the rateof wound healing and the proportion of wounds that heal in diabeticanimals and in humans. Treatment with insulin also increased expressionof eNOS, VEGF, and SDF-lalpha in wounded skin. Rezvani conducted an RCTin diabetic foot wounds to evaluate topical insulin on healing in 45patients. The mean rate of healing was 46.09 mm²/day in the treatmentgroup, and 32.24 mm²/day in the control group (p=0.03). These datasuggest that insulin can improve wound healing and may be beneficialwhen used in an in vitro model to increase cell proliferation and wouldenhance cell proliferation into the collagen matrix.

Example 3.3

3-4 days following the first application of autologous cells, and as theallogeneic cells and matrix begin to form obvious healthy epithelialtissue, lyophilized amniotic membrane (AM) is sprayed (such as from amodified airbrush-like apparatus (preferably associated with the printhead of the 3D printer) onto the cell-seeded bovine collagen. There is anotable body of evidence to suggest that freeze-dried, powdered amnioticmembrane promotes rapid healing and enhances the “take” rate of grafts.AM also inhibits natural inflammatory reactions which contribute tohealthy tissue adhesion and structural development. There is evidence tosuggest that combined with an electrical field, the application of AMwill enhance cell migration and angiogenesis to cells located in thecenter-most region of the graft bed.

Example 3.4

Continual layers of the cultured material are printed onto collagenplates until desired thickness is achieved. Amount of cells wanted ineach layer, number of times the printer must create layers for the skingraft, intervals between applications, and types and amounts of growthfactors and other ECM proteins to be added are factors.

EXAMPLE 4

Multiple copies of the autograft are printed. (In this example, multiplecopies are printed. It will be appreciated that in other cases due tolimited donor site material there will only be enough to print onecopy.) The first is transplanted to the primary wound within 5-7 days.During the 5-7 days preparation period, negative pressure wound therapywith or without simultaneous irrigation (e.g., saline) is applied toprepare the wound bed for graft acceptance as well as reduce bacterialload. Negative pressure therapy is known to induce angiogenesis and thisincrease in blood flow and the resultant delivery of nutrients not onlyto the wound bed but to the newly placed engineered craft is critical toits survival and success.

As was described hereinabove in the Background with respect to severalstudies conducted using amniotic membrane (AM) in both acute and chronicwounds, much of the first round placement was absorbed into the body. Insome cases, it took as many of 3-4 full grafts of AM in order to resultin full closure of the wound when using that conventional technology. Bycontrast, with skin printing according to the invention, a much thickerand partially autologous engineered graft that more closely approximatesnatural human skin is provided. A thicker, partially autologousengineered graft has improved probability of survival and ability tomake active contributions to recruiting the active mechanisms ofhealing. Meanwhile, in practicing the invention, the additional skingrafts continue to mature and if necessary, are useable as the finalstep to closure. In the alternative, the graft copies could be stored ina tissue bank for later use by the same patient if, for example,additional surgical revisions were anticipated.

While the invention has been described in terms of a preferredembodiment, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

1-36. (canceled)
 37. A skin graft product constructed from skin cells ofa patient having a wound, wherein an amount of patient skin cells isless than the patient skin cells that would be estimated to be needed totreat the wound if only the patient skin cells were used.
 38. The skingraft product of claim 37, consisting of: an amount of patient skincells which is less than the patient skin cells that would be estimatedto be needed to treat the wound by conventional skin grafting if onlythe patient skin cells were used; an amount of material other thanpatient skin cells.
 39. The skin graft product of claim 37, wherein theamount of patient skin cells is selected from the group consisting of:about ⅔ what would be estimated to be needed to treat the wound if onlythe patient skin cells were used; less than ⅔ what would be estimated tobe needed to treat the wound if only the patient skin cells were used;less than ½ what would be estimated to be needed to treat the wound ifonly the patient skin cells were used; less than ⅓ what would beestimated to be needed to treat the wound if only the patient skin cellswere used; less than ¼ what would be estimated to be needed to treat thewound if only the patient skin cells were used; and less than ⅕ whatwould be estimated to be needed to treat the wound if only the patientskin cells were used.
 40. The skin graft product of claim 38, whereinthe amount of material other than patient skin cells comprises one ormore of bovine collagen, growth factors, amniotic membrane andcytokines. 41-53. (canceled)